CN106539758A - A kind of non-vesicle type nano particle of high stable and its application in treatment infection of staphylococcus aureus - Google Patents
A kind of non-vesicle type nano particle of high stable and its application in treatment infection of staphylococcus aureus Download PDFInfo
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Abstract
The non-vesicle type nano particle that the present invention provides a kind of aliphatic acid by C16-C20, surfactant and optional lipid are constituted.The non-vesicle type nano particle of the present invention is directed to staphylococcus, particularly staphylococcus aureus, and preferred methicillin-resistant staphylococcus aureus (MRSA) possesses significant antibacterial activity；The non-vesicle type nano particle of the present invention is also equipped with excellent stability, and the not generation of inducible resistance strain such that it is able to used as outstanding antibacterials.
Description
Technical field
The present invention relates to drug world.Specifically, the present invention relates to a kind of high stable, non-vesicle type receives
Rice grain, and this nano particle is in treatment staphylococcus (Staphylococcus), particularly golden yellow Portugal
Grape coccus (Staphylococcus aureus), preferred methicillin-resistant staphylococcus aureus
Application in (Methicillin-resistant Staphylococcus aureus, MRSA) infection.
Background technology
Staphylococcus, particularly staphylococcus aureus (Staphylococcus aureus) are that a kind of initiation causes
The nosocomial infection of life property and the one of the main reasons of Nosocomial Infections.In recent decades, staphylococcus aureus
Experienced several ripples and there is the resistance to the action of a drug to the opposing of antibiotic and now to the antibiotic of whole beta-lactam class, wrap
Include PCs, cephalosporin class and carbapenem antibiotic.
It is resistance to that these have drug-fast staphylococcus aureus also referred to as methicillin to beta-lactam class antibiosis
Medicine staphylococcus aureus (MRSA), or superbacteria.MRSA infection increasingly increase result in through the ages
Mycin is commonly used, and vancomycin is only still to one of effective antibiotic of MRSA at present.But
Vancomycin can only also suppress MRSA but eradicate them.Additionally, since nineteen ninety is for the later stage, through the ages
The a large amount of of mycin use the appearance that result in vancomycin resistance staphylococcus aureus strain.All these is true
The importance and urgency of demand to developing new effectively treatment MRSA method have been highlighted all.
Drug research with regard to new anti-MRSA synthesizes new drug on the basis of being mainly included in existing antibiotic recently
Thing, exploitation immunoglobulin (Ig) remove MRSA toxin to target, and develop some natural antibiotics, for example sun
Ion antibacterial peptide, liposome and other endogenous things.Although these new synthetic drugs achieve good clinic
Result of the test, but they possess similar structure and Antibacterial Mechanism to existing antibiotic, therefore, in synthesis
Class medicine is by soon, MRSA will develop immunity to drugs to them after extensive Clinical practice.It is antitoxin by injecting
Globulin and antibody fragment come passively neutralize the bacteriotoxin factor may to improve acute infection be it is beneficial, but
It is that this method can not suppress or eradicate bacterium itself.And In the view of for a long time, recurrence still can be caused
Sexy dye.
In the past few decades, application of the nanometer technology in materia medica is widely explored.By physics bag
Cover or chemical bond, medicine can be loaded into nano particle, so as to compared to free drug, significantly improve
The kinetic index and therapeutic index of medicine.Collecting these advantages based on the delivery system of nano particle more
In：Improve medicine serum solubility, extend medicine the body circulation cycle and continue, controllably discharge
The aspects such as medicine.As the medicine in these nano particles mostly is traditional antibiotic, resistance still can be produced
Strain.And, the nano particle preparation process for being used is complicated, high cost, and stability is limited, so as to
The actual application value of this kind of medicine is had a strong impact on.
Therefore, this area be badly in need of it is efficient, do not induce produce persister to resist staphylococcus, particularly gold
The new types of therapeutic agents of staphylococcus aureus, more preferably methicillin-resistant staphylococcus aureus infection.
The content of the invention
It is an object of the invention to provide a kind of staphylococcus of resisting that is efficient, not inducing generation persister, special
It is not staphylococcus aureus, the novel therapeutic medicine that more preferably methicillin-resistant staphylococcus aureus infects
Thing.
In a first aspect, a kind of non-vesicle type nano particle of present invention offer, the particle is by aliphatic acid, surface
Activating agent and optional lipid are constituted, wherein, the aliphatic acid is C16-C20, the fat of preferred C17-C19
Acid.
In a preferred embodiment, the aliphatic acid possesses two or more unsaturated bonds.
In a particular embodiment, the aliphatic acid is leukotrienes.
In a preferred embodiment, the surfactant includes but is not limited to following one or more：Firmly
Resin acid sodium, 4- (5- dodecyls) benzene sulfonate, polyoxyethylene glycol, polysorbate 20, polyoxyethylene sorbitan monoleate,
Polysorbas20, Tween 80 and Triton X-100.
In a preferred embodiment, the lipid is phosphatide and/or cholesterol.
In a preferred embodiment, the phosphatide includes but is not limited to following one or more：Phosphatidyl second
Hydramine, phosphatidylinositols, phosphatidylserine, phosphatid ylcholine, two myristoyl lecithin, two palmityls
The oily phosphatidyl-ethanolamine of phosphatid ylcholine, palmityl phosphatidyl glycerol and two.
In a preferred embodiment, the mass ratio of the lipid and surfactant is 10～0:1；It is preferred that 5～0:
1；More preferably 2.5～0:1.
In a particular embodiment, the particle diameter of the non-vesicle type nano particle is 1-90nm；It is preferred that 2-80nm；
More preferably 5-50nm；More preferably 5-20nm；Most preferably 5-15nm.
In a preferred embodiment, the particle diameter of the nano particle can be 1-30nm；10-40nm；20-50
nm；30-60nm；40-70nm；50-80nm；60-90nm；Or, the particle diameter of the nano particle can be with
For 5-25nm；15-35nm；25-45nm；35-55nm；45-65nm；55-75nm；65-85nm or,
The particle diameter of the nano particle can be 10-30nm；20-40nm；30-50nm；40-60nm；50-70nm；
60-80nm；70-90nm.
In a particular embodiment, the polydispersity coefficient of the nano particle is<0.3；It is preferred that<0.2.
In a particular embodiment, the stability of the non-vesicle type nano particle is：
After preserving 2 months under room temperature, compared with the nano particle of fresh preparation, the nano particle is directed to MRSA
" minimal inhibitory concentration " and " MBC " value change be less than 20%；Preferably smaller than 10%；Or
Person；
Or, change in the particle diameter of 4 DEG C, the 25 DEG C and 37 DEG C storages nano particle of the present invention of 3 months and be less than
20%；It is preferred that the particle diameter of nano particle of the present invention changes less than 15%；The grain of nano particle more preferably of the present invention
Footpath changes less than 10%.
In a preferred embodiment, the nano particle is directed to 1 × 106The MIC value of the MRSA252 of CFU is
0.1%w/v；The nano particle is directed to 1 × 106The MBC values of the MRSA252 of CFU are 0.2%w/v.
In a preferred embodiment, the concentration of the aliphatic acid is 0.1～4%w/v；It is preferred that 0.2～3%w/v；More
It is preferred that 0.3～2%w/v.
In a particular embodiment, the nano particle is prepared by following methods, and methods described includes：
1). by surfactant and optional lipid suspension in water；
2). the suspension that 1) stirring obtains is until form homogeneous suspension liquid；
3). it is more than the fusing point of surfactant and optional lipid contained by the homogeneous suspension liquid to its that 2) heating obtains；
4). aliphatic acid is added in the hot suspension for 3) obtaining and stirred；
5). cooling, the suspension for 4) obtaining is stood, so as to obtain the non-vesicle type nano granule suspension of the present invention.
In a particular embodiment, nano particle of the invention is used to prepare antistaphylohemolysin
(Staphylococcus) reagent for infecting, or can be used to treat staphy lococcus infection；Preferably, the staphylococcus
It is staphylococcus aureus (Staphylococcus aureus)；More preferably methicillin-resistant staphylococcus aureus
(Methicillin-resistant Staphylococcus aureus)。
In second aspect, the present invention provides the preparation side of the non-vesicle type nano particle described in first aspect present invention
Method, comprises the following steps：
1). by surfactant and optional lipid suspension in water；
2). the suspension that 1) stirring obtains is until form homogeneous suspension liquid；
3). it is more than the fusing point of surfactant and optional lipid contained by the homogeneous suspension liquid to its that 2) heating obtains；
4). aliphatic acid is added in the hot suspension for 3) obtaining and stirred；
5). cooling, the suspension for 4) obtaining is stood, so as to obtain the non-vesica any one of claim 1-5
Type nano particle.
In a preferred embodiment, the melting temperature in methods described be 20 DEG C -80 DEG C, such as 20 DEG C, 30 DEG C,
40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C or 80 DEG C.
In a preferred embodiment, methods described may also include the hydrodynamics chi of detection gained nano particle
It is very little.
In a preferred embodiment, the aliphatic acid is C16-C20, the aliphatic acid of preferred C17-C19.
In a preferred embodiment, the aliphatic acid is leukotrienes.
In a preferred embodiment, the lipid is phosphatide and/or cholesterol.
In a preferred embodiment, the phosphatide includes but is not limited to following one or more：Phosphatidyl second
Hydramine, phosphatidylinositols, phosphatidylserine, phosphatid ylcholine, two myristoyl lecithin, two palmityls
The oily phosphatidyl-ethanolamine of phosphatid ylcholine, palmityl phosphatidyl glycerol and two.
In a preferred embodiment, the proportioning of the lipid and surfactant is 10～0:1；It is preferred that 5～0:1；
More preferably 2.5～0:1.
In a preferred embodiment, the surfactant is included including but not limited to following one or more：
Odium stearate, 4- (5- dodecyls) benzene sulfonate, polyoxyethylene glycol, polysorbate 20, polyoxyethylene sorbitan monoleate,
Polysorbas20, Tween 80 and Triton X-100.
In a preferred embodiment, the concentration of the aliphatic acid is 0.1～4%w/v；It is preferred that 0.2～3%w/v；More
It is preferred that 0.3～2%w/v.
In a preferred embodiment, the particle diameter of obtained nano particle is 1-90nm；It is preferred that 2-80nm；It is more excellent
Select 5-50nm；More preferably 5-20nm；Most preferably 5-15nm.
In a preferred embodiment, the particle diameter of obtained nano particle can be 1-30nm；10-40nm；20-50
nm；30-60nm；40-70nm；50-80nm；60-90nm；Or, the particle diameter of the nano particle can be with
For 5-25nm；15-35nm；25-45nm；35-55nm；45-65nm；55-75nm；65-85nm or,
The particle diameter of the nano particle can be 10-30nm；20-40nm；30-50nm；40-60nm；50-70nm；
60-80nm；70-90nm.
In a preferred embodiment, the polydispersity coefficient of obtained nano particle is<0.3；It is preferred that<0.2.
In a preferred embodiment, the stability of obtained nano particle is：
After preserving 2 months under room temperature, the MIC and MBC value of the nano particle is equal to the nanometer of fresh preparation
Grain；Or,
Change less than 20% in the particle diameter of 4 DEG C, the 25 DEG C and 37 DEG C storages nano particle of the present invention of 3 months；It is excellent
The particle diameter of anthology invention nano particle changes less than 15%；The particle diameter of nano particle more preferably of the present invention changes little
In 10%.
In the third aspect, the present invention provides a kind of pharmaceutical composition, and which includes non-described in first aspect present invention
Vesicle type nano particle and optional pharmaceutically acceptable carrier.
In a preferred embodiment, the formulation of described pharmaceutical composition is included but is not limited to：Suitable general is given
The formulation of medicine or the formulation of external application or local administration；
Further, the formulation is included but is not limited to：Tablet, solution, suspension, capsule,
Granula, pulvis, injection, patch, spray, ointment, ointment, cream, gel, creme,
Drops, spray, lotion；
It is preferred that the formulation of external application or local administration, including but not limited to：Patch, spray, ointment, ointment,
Cream, gel, creme, drops, spray, lotion.
In a preferred embodiment, the pharmaceutically acceptable carrier is included but is not limited to：Water；Physiology
Salt solution；Bonding agent (such as polyvinylpyrrolidone or hydroxypropyl methyl cellulose)；Filler (for example lactose and
Other carbohydrates, gelatin or calcium sulfate)；Lubricant (such as starch, polyethylene glycol or sodium acetate)；Disintegrant (example
Such as starch or sodium starch glycollate)；And wetting agent (such as NaLS).
In a preferred embodiment, described pharmaceutical composition can also contain penetration enhancers；It is described to penetrate rush
Enter agent to include but is not limited to：Surfactant (such as lauryl sodium sulfate, laureth9
With polyoxyethylene -20- cetyl ethers)；Bile salt (such as cholic acid, dehydrocholic acid and deoxycholic acid)；Chela
Mixture (such as disodium ethylene diamine tetraacetate, citric acid and salicylate)；And non-chelated property on-surface-active
Agent (such as unsaturation ring urea).
In a particular embodiment, described pharmaceutical composition can also include other antibiotic.
In a preferred embodiment, the antibiotic is to treat staphylococcus, preferred staphylococcus aureus
(Staphylococcus aureus)；More preferably methicillin-resistant staphylococcus aureus (Methicillin-resistant
Staphylococcus aureus) antibiotic that infects, including but not limited to：Vancomycin, cynnematin, profit
How azoles amine, teicoplanin, Arbekacin, quinupristin, Dalfopristin, quinoline Knoop fourth, clindamycin,
Daptomycin, rifampin, for drawing ten thousand stars, the medicine such as Tetracyclines such as tigecycline.
In a particular embodiment, described pharmaceutical composition is aqueous pharmaceutical compositions.
In fourth aspect, the present invention provides the medicine of the nano particle or third aspect present invention of first aspect present invention
Purposes of the compositions in antistaphylohemolysin (Staphylococcus) reagent is prepared.
In a particular embodiment, the staphylococcus is staphylococcus aureus (Staphylococcus
aureus)；More preferably methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus
aureus)。
In terms of the 5th, the present invention provides a kind for the treatment of method, and the treatment method is included first party of the present invention
Nano particle described in face gives object to treat microorganism infection.
In another embodiment, the treatment method include by the nano particle described in first aspect present invention with
Other Antibiotic combinations give object to treat microorganism infection.
In a particular embodiment, the staphylococcus is staphylococcus aureus；More preferably methicillin is resistance to
Medicine staphylococcus aureus.
In a preferred embodiment, described other antibiotic are to treat staphylococcus, preferred Staphylococcus aureus
Bacterium (Staphylococcus aureus)；More preferably methicillin-resistant staphylococcus aureus
The antibiotic that (Methicillin-resistant Staphylococcus aureus) infects, including but not limited to：It is mould through the ages
Element, cynnematin, Linezolid, teicoplanin, Arbekacin, quinupristin, Dalfopristin, quinoline are exerted
General fourth, clindamycin, Daptomycin, rifampin, for drawing ten thousand stars, the medicine such as Tetracyclines such as tigecycline；
The antibiotic can also treat the antibiotic that other type of micro-organisms infect, such as quinolones, β-interior acyl
Amine, macrolides, aminoglycosides, amphenicols, nitro glyoxaline etc..
In a preferred embodiment, the nano particle utilizes identical or different administration way with other antibiotic
Footpath, gives in identical or different administration time.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (such as embodiment)
Can be combined with each other between each technical characteristic of middle specific descriptions, so as to constitute new or preferred technical side
Case.As space is limited, here is no longer tired out one by one and is stated.
Description of the drawings
Fig. 1 is the photo of one group of nano particle of the present invention, and wherein linolenic acid concentration is from the 0.1%w/v in (a) to (h)
In 4%w/v (wherein %w/v be equal to wt%).Concentration class and clarity according to these samples etc. is physical
Matter, it may be determined that preferable nano particle formulation.
Fig. 2 shows the long-time stability containing the linolenic nano particles of the present invention of 1%w/v, by 3 months
Time span in detect their hydrodynamic diameter.In this time span, these nano particles
4 DEG C, 25 DEG C and 37 DEG C are stored in respectively, diameter but increases less than 2nm, illustrated that the height of this formulation is steady
It is qualitative.
Fig. 3 shows external minimal inhibitory concentration (MIC) of the nano particle of the present invention to MRSA252.It is different
The nano particle of concentration with MRSA252 (1*106CFU/mL)(CFU：CFU) train together
Educate, trap of the bacterium under OD600 was measured at 5 hours and 24 hours respectively.As a result show nanometer of the present invention
Particle can suppress the growth of bacterium in the case where concentration is higher than 0.1%w/v or higher.5 hours and 24 hours
Bacterium and the suspension mixed picture of nano particle of the present invention show that the solution of 0.1%w/v nano particles is limpid
It is bright, show that 0.1%w/v is the MIC of nano particle of the present invention.
Fig. 4 shows external MBC (MBC) of the nano particle of the present invention to MRSA252.It is different
The nano particle of the present invention of concentration and MRSA252 (1*106CFU/mL) incubation 24 hours, then 5
The suspension of μ L is overnight at 37 DEG C to be cultivated and observed on TSB agar plates.The CFU of MRSA252 is worth
To quantify.(A) image shows that the nano particle of the present invention and MRSA252 of variable concentrations are educated on a lbmc agar plate altogether
The CFU of the MRSA252 observed after 24 hours；(B) result shows that the nano particle of the present invention of 0.2%w/v is killed
99.9% MRSA252 in the dust.In addition bacterium reaches 0.4%w/v or more in the concentration of nano particle of the present invention
All killed when high.
Fig. 5 shows forms of the MRSA252 after the form (A) of nano particle before processing of the present invention and process
(B).In (B), bacterium is imaged after having been cultivated 24 hours with nano particle of the present invention.In all of experiment,
The initial concentration of bacterium is all 1*106CFU/mL.Engineer's scale in picture is 1 μm.
Fig. 6 shows the long-term antimicrobial activity of nano particle of the present invention.Nano particle of the present invention is protected at room temperature
Deposit.Storage
Sample MIC after 3 months slightly brings up to 0.2%w/v, and MBC values then with it is consistent before.
Fig. 7 shows antibacterial activity in vivo of the nano particle of the present invention to MRSA252.Mouse is infected
1*107The MRSA252 of CFU.Then the gel containing nano particle of the present invention is used once daily, and continuous 5
My god.Carry bacterium amount MRSA252 be inoculated with 5 days after, the skin infection with mouse be removed come, homogenizing and
Cultivate on agar plate, to record bacterium CFU.Tables of data understands six mean+SDs individually tested.
* conspicuousness (the * * p of p value are represented<0.01)
Fig. 8 shows antibacterial activity in vivo of the nano particle of the present invention to the mouse of MRSA252 subcutaneous infections.
In test, mouse has been subcutaneously injected into 1*106The MRSA252 of CFU, subsequently after same position 20 minutes again
Nano particle of the present invention is injected.After picture shows that injection is in MRSA injections 24,48 and 72 hours,
The situation of damage location.
Fig. 9 shows toxicity in vivo researchs in 7 days of nano particle external-use gel formulation of the present invention.Beaten with Draize
Subsystem shows that the use of nano particle formulation of the present invention does not produce obvious oedema or erythema.Image is every
The representative of 5 mouse of group.
Figure 10 shows the evaluation result of H＆E (left column) and TUNEL (right row) to assess nanometer of the present invention
The toxicity in vivo of grain.Blank PBS gels are used as negative control group.Nano particle formulation of the present invention does not have
Produce inflammation and no obvious cell death.Image is the representative of per group of 5 mouse.
Figure 11 shows use of the macrophage to evaluation result-assessment nano particle of the present invention of skin infiltration
Security.After the frozen section of skin is prepared, with DAPI to nuclear targeting, FITC- anti-mouse is used
F4/80 antibody is dyeed to skin macrophage.After dyeing, dermatological specimens use Nikon Delta immediately
Macroview fluorescence microscopes are imaged.Image is the representative of per group of 5 mouse.Engineer's scale in picture is 400
μm。
Figure 12 shows the structural representation of nano particle of the present invention.
Figure 13 shows the size distribution curve of nano particle of the present invention.
Figure 14 compared for nano particle of the present invention and MBC of the free fatty in aqueous environments.
Figure 15 shows bactericidal activity of the nano particle of the present invention comprising different aliphatic acid to MRSA.
Specific embodiment
Inventor through extensively in-depth study, it was unexpectedly found that by aliphatic acid, surfactant with
And non-vesicle type nano particle (micellar nanoparticles structure) not only possesses significant antibacterial made by optional lipid
Activity, moreover it is possible to possess excellent stability, and the product of the non-vesicle type nano particle of the present invention not inducible resistance strain
It is raw such that it is able to as outstanding antibacterials.The present invention is completed on this basis.
Non- vesicle type nano particle
Term used herein, " nano particle ", " nano particle of the present invention ", " non-vesicle type nanometer
Particle " and " the non-vesicle type nano particle of the present invention " possess identical implication, each mean
The nano particle of alveolitoid formula.Specifically, non-vesicle type nano particle of the invention is relative to other tools
For having the nano particle of cavity structure, i.e. the non-vesicle type nano particle of the present invention is that wherein do not possess
The nano particle of cavity.Additionally, the preparation method based on invention described below nano particle, this area
Technical staff is appreciated that the nano particle of the present invention is a kind of nanoparticle system, i.e. in aqueous body
Nano particle in system, or the water-based system entirety comprising nano particle；In other words, it is of the present invention to receive
Rice grain is a kind of water-based system of nano particle, i.e. a kind of nanoparticle system without organic solvent.
The non-vesicle type nano particle that the present invention is provided is made up of aliphatic acid, surfactant and optional lipid.
The molecule of these constituents all has the hydrophobic part of the hydrocarbon chain composition of hydrophilic part and an elongation.
For example, the amphiphilic that aliphatic acid (such as leukotrienes) is made up of a hydrophobic hydrocarbon chain and hydrophilic carboxylic acid head group
Property molecule, this structure enable aliphatic acid include nano-micelle so with amphipathic environment nanometer
In grain.In presence of water, their hydrophilic segment and surfactant are queued up and define one facing to water
Surface, at the same time hydrophobic part queue up define a core away from water, so as to define micella
Nanostructured (as shown in figure 12).
In a particular embodiment, the aliphatic acid in non-vesicle type nano particle of the invention is the fat of C16-C20
Fat acid.In a preferred embodiment, aliphatic acid is the aliphatic acid of C17-C19.In a preferred embodiment,
Aliphatic acid used by of the invention is comprising multiple, such as aliphatic acid of 2-4 unsaturated double-bond.Further preferred
In embodiment, the aliphatic acid is leukotrienes (C18)." leukotrienes " as herein described contains this fat
The acid of acid, three kinds of forms of salt and ester.
In a particular embodiment, the lipid be phosphatide, including but not limited to following one or more：
Phosphatidyl-ethanolamine, phosphatidylinositols, phosphatidylserine, phosphatid ylcholine, two myristoyl lecithin,
The oily phosphatidyl-ethanolamine of DPPC, palmityl phosphatidyl glycerol and two.
In a particular embodiment, the surfactant includes but is not limited to following one or more：Firmly
Resin acid sodium, 4- (5- dodecyls) benzene sulfonate, polyoxyethylene glycol, polysorbate 20, polyoxyethylene sorbitan monoleate,
Polysorbas20, Tween 80 and Triton X-100.
In a particular embodiment, the excipient materials of nano particle of the invention can comprising phosphatid ylcholine,
Cholesterol, lecithin, polysorbas20, Tween 80 and lauryl sodium sulfate.
In the non-vesicle type nano particle of the present invention, the proportioning of lipid and surfactant is 10～0:1；It is preferred that
5～0:1；More preferably 2.5～0:1.
The non-vesicle type Nanoparticulate composition of the present invention includes appropriate aliphatic acid, preferably linolenic acid.This composition
Effectively can suppress to have cultivated the gold of 5 hours at 37 DEG C in soybean trypsin culture medium (TSB)
The growth of staphylococcus aureus.In some embodiments, nano particle of the invention includes at least 0.1
The aliphatic acid of mg/mL, such as leukotrienes (for example, at least 1mg/mL, at least 10mg/mL, at least 20
Mg/mL, at least 30mg/mL, at least 50mg/mL or at least 100mg/mL).In specific embodiment
In, the concentration of the aliphatic acid is 0.1～4%w/v；It is preferred that 0.2～3%w/v；More preferably 0.3～2%w/v.Excellent
In the embodiment of choosing, aliphatic acid in the nano particle of the present invention, such as linolenic acid content are 1.0%w/v.
It will be understood by those skilled in the art that aliphatic acid as herein described, such as linolenic concentration refers to the fat
Acid is in the system comprising nano particle of the present invention, particularly water-based system, such as concentration of waterborne suspension.
The non-vesicle type nano particle of the present invention possesses a series of physicochemical characteristicses, the diameter of such as nano particle
With linolenic concentration (that is, mass percent of the leukotrienes in whole nano granule suspension).Nanometer
The diameter of grain can be measured using dynamic light scattering method.The non-vesicle type nano particle of the present invention it is average straight
Footpath is about 1 to 90nm；It is preferred that 2-80nm；More preferably 5-50nm；More preferably 5-20nm；Most preferably 5-15nm.
In other embodiments, the particle diameter of the nano particle can be 1-30nm；10-40nm；20-50nm；
30-60nm；40-70nm；50-80nm；60-90nm；Or, the particle diameter of the nano particle can be 5-25
nm；15-35nm；25-45nm；35-55nm；45-65nm；55-75nm；65-85nm or, it is described
The particle diameter of nano particle can be 10-30nm；20-40nm；30-50nm；40-60nm；50-70nm；60-80
nm；70-90nm.
The particle diameter distribution of the non-vesicle type nano particle of the present invention is homogeneous, and its polydispersity coefficient is<0.3；It is preferred that
<0.2.The size distribution curve of the non-vesicle type nano particle of the present invention is as shown in figure 13.
The non-vesicle type nano particle of the present invention possesses excellent stability.In a particular embodiment, in room
After the lower preservation of temperature 2 months, the MIC and MBC value of nano particle of the present invention is equal to the nano particle of fresh preparation；
Store 3 months under room temperature, nano particle of the present invention is directed to 1 × 106The MIC value of the MRSA252 of CFU compared to
The nano particle of fresh preparation only increases to 0.2%w/v, and MBC values are constant.In another embodiment, 4
DEG C, 25 DEG C and 37 DEG C storage 3 months nano particles of the present invention particle diameter change be less than 20%；Preferably, originally
The particle diameter of invention nano particle changes less than 15%；It is further preferred that the particle diameter of nano particle of the present invention changes being less than
10%.
The non-vesicle type nano particle of the present invention possesses excellent antibacterial activity.In a particular embodiment, originally
The nano particle of invention can suppress or kill staphylococcus (Staphylococcus).In a preferred embodiment,
The staphylococcus is staphylococcus aureus (Staphylococcus aureus).In preferred embodiment,
The staphylococcus is methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus
aureus)。
In a particular embodiment, nano particle of the invention is directed to 1 × 106The MRSA minimums of CFU are antibacterial
Concentration (MIC) is 0.1%w/v, and MBC (MBC) is 0.2%w/v.
The nano particle of the present invention suppresses the ability of staphylococcus aureus determine as follows.For example, by gold
It is incubated in staphylococcus aureus and nano particle of the invention in vitro culture medium altogether, or in animal model
It is incubated altogether.Then measure the clump count (being characterized as CFU) of staphylococcus aureus.Terms used herein " suppression
System growth " suppresses the ability that CFU increases when referring to that the nano particle of the present invention and staphylococcus aureus educate altogether.
Therefore, when staphylococcus aureus is contacted with the nano particle of the present invention, the CFU of staphylococcus aureus
Will not change or can decline.
Terms used herein " minimal inhibitory concentration " is (MIC) to refer to suppress bacterium, such as golden yellow
The medicine of aureus growth, such as Cmin of nano particle of the present invention.MIC value can be by measurement
Optical density of the staphylococcus aureus after the nano particle of the present invention for contacting various concentration determining, especially
Whether relatively OD600 values change.After a period of time, if OD600 is not raised, show
Bacterial number in nutrient solution is not raised.
Terms used herein " MBC " (MBC) be refer to kill bacterial cell medicine,
The least concentration of such as nano particle of the present invention.MBC can by, for example, count certain specified conditions under
The colony growth situation on culture medium that the particle of the present invention of (such as 37 DEG C) and variable concentrations is educated altogether is determining.
The preparation method of the non-vesicle type nano particle of the present invention
The non-vesicle type nano particle of the present invention can be prepared using following methods, comprised the following steps：
1). by surfactant and optional lipid suspension in water；
2). the suspension that 1) stirring obtains is until form homogeneous suspension liquid；
3). it is more than the fusing point of surfactant and optional lipid contained by the homogeneous suspension liquid to its that 2) heating obtains；
4). aliphatic acid is added in the hot suspension for 3) obtaining and stirred；
5). cooling, the suspension for 4) obtaining is stood, so as to obtain the non-vesicle type nano granule suspension of the present invention.
In a particular embodiment, the melting temperature in methods described be 20 DEG C -80 DEG C, such as 20 DEG C, 30 DEG C,
40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C or 80 DEG C.
In further embodiment, methods described may also include the hydrodynamics of detection gained nano particle
Size.
The preparation of the non-vesicle type nano particle of the present invention needs to consider following parameter, for example raw material and will be by
The physicochemical properties of the material being included in nano particle, for the property of the medium of dispersing nanoparticles,
By the valid density of loading matter and his genotoxic potential, the process involved by nano particle is applied/transports,
Most suitable size, poly dispersiveness and shelf-life, batch reproducibility and large-scale production safety and high efficiency products
Possibility.
The preparation of the nano particle of the present invention is not what is spontaneously formed, is provided in feedwater when there is enough energy
Aliphatic acid, for example leukotrienes and could be formed during excipient materials (for example by ultrasonically treated, homogenizing,
Vibration is heated).
The nano particle of the present invention can be prepared by various compositions provided by the present invention and method.It is applied to
Other preparation methods of nano particle of the present invention include paddling process and heating, including high-speed stirred.This skill
The advantage of art is that equipment is simple, and production is easy.
In some embodiments, the nano particle can be formed by high-pressure homogenization.High-pressure homogenization
It is widely used in various industries, it is considered to be most viable industry application method thereof.This technology contains and is being higher than
Or it is low prepare Solid lipid nano particle at room temperature, cavitation erosion and eddy current effect reduce can particle size.Make
Melt can liposome and medicine with thermal high homogenate method, and at equal temperature with aqueous surfactant solution
Combination.The pre- emulsion of heat is processed in the high pressure homogenizer of temperature control again, typically will be in the bar of 500bar
Most 3 circulations are carried out under part.The nanoemulsion for being obtained is recrystallized after cooling to room temperature, forms solid
State elaioplast nanometer particle.Cold anticyclone homogenate method can be used to process hydrophilic medicament.
In addition to the method described above, other any methods for preparing solid lipids nano particle can
For producing the nano particle of the present invention.Such method includes microemulsion method, emulsification-evaporation method, emulsification
Solvent diffusion method, solvent injection method and phase inversion method.
The nano particle that obtains of preparation method of the present invention possesses excellent steady in structure and BA
It is qualitative；The preparation method of the present invention is also very abundant to the utilization rate of aliphatic acid.
Pharmaceutical composition and its using method
The nano particle of the present invention can make pharmaceutical composition for use in people and other mammals.Use
When can mix with other medicines carrier or diluent, and according to the feature of the mammal state of an illness
Dosage and the cycle of administration are determined with seriousness.Under normal circumstances, the aliphatic acid in nano particle of the present invention,
For example leukotrienes should be able to reach pharmacy effective dose；For example, depositing on mammal skin can effectively be reduced
The dosage of staphylococcus aureus quantity living.
Preparation and medication are all known to industry veteran.Under normal circumstances, according to being controlled
The seriousness of disease condition reacts to be administered with drug effect, course for the treatment of last for days to several months, until disease amelioration is
Only.Optimal dosage, medication and repetitive rate are also that this area ordinary people can determine.Most preferably
Dosage can be adjusted according to the relative efficacy of nano particle of the present invention, typically can be according to external and body
MIC the and MBC values of interior animal model are measured using effect to estimate.Administration frequency can be once a day or many
Secondary, biweekly, the weekly or longer time is once.After treatment success, some maintenances should be proceeded
Treatment, in case infection and recurrence.
Term " pharmaceutically acceptable carrier " used herein possesses identical implication with " excipient ",
Each mean for the solvent to the pharmacy accreditation that nano particle of the present invention is conveyed using object, suspending agent or any
The excipient of other pharmaceutical inerts.Pharmaceutical acceptable carrier can be liquid or solid, should be according to being planned
Administering mode selecting carrier, so as to when the nano particle and one or more medicativeization of the present invention
When compound or other Pharmaceutical Compositions are combined, preferable dosage, uniformity and other drug deliveries can be reached
With the characteristic of chemistry.
Adverse reaction will not be produced with the nano particle of the present invention or destroy the nano junction of nano particle of the present invention
The pharmaceutical acceptable carrier of structure is included but is not limited to：Water；Physiological saline；Bonding agent (such as polyvinyl pyrrole
Alkanone or hydroxypropyl methyl cellulose)；Filler (such as lactose and other carbohydrates, gelatin or calcium sulfate)；Profit
Lubrication prescription (such as starch, polyethylene glycol or sodium acetate)；Disintegrant (such as starch or sodium starch glycollate)；
And wetting agent (such as NaLS).
The nano particle of the present invention can be administered using various methods, but typically local is administered.The present invention's
Nano particle can be with other molecular mixings, or the mixture with other molecules, molecular structure or compound
Be used together, for example polyethylene glycol, vaseline, or other external preparations, so as to promote medicine intake,
Distribution and/or absorption.The formulation of local administration may include aseptic and non-sterile aqueous solutions, and common solvent
Non-aqueous solution (such as ethanol or liquid or solid oil matrix solution).Such solution can also containing buffer solution,
Diluent and other suitable additives.Local administration pharmaceutical dosage form include transdermal patch, ointment, lotion,
Creme, gel, drops, spray, liquid and powder；Wherein particularly preferred lotion, creme and gel
Agent.Chang Keneng needs Conventional pharmaceutical carriers (water-based, powdered substrate or oil matrix) when in use,
It is likely to use the other materials such as thickener.In some cases, the nano particle of the present invention may suspend
In suspension in water-based, non-aqueous matrix or mixed-matrix.Can also suspend containing increasing in suspension
The material of fluid viscosity, such as sodium carboxymethylcellulose, sorbierite and/or glucan.Suspension can also be included
Stabilizer.
In a preferred embodiment, pharmaceutical composition of the invention can also contain penetration enhancers to improve this
Invention nano particle effectively penetrates the function of mammal skin.Penetration enhancers can improve simultaneously lipophilicity with
Ability of the non-lipophilic drugs through cell membrane.Penetration enhancers are included but is not limited to：Surfactant (example
Such as lauryl sodium sulfate, laureth9 and polyoxyethylene -20- cetyl ethers)；Bile acid
Salt (such as cholic acid, dehydrocholic acid and deoxycholic acid)；Chelating agent (such as disodium ethylene diamine tetraacetate, lemon
Acid and salicylate)；And non-chelated property non-surface-active agent (such as unsaturation ring urea).
In addition, in some embodiments, the nano particle of the present invention can be passed by iontherapy
Send, using the transdermal patch with electric charge, come " ordering about ", the nano particle reaches corium.
Some other ancillary drug compositions can generally also be contained in the pharmaceutical composition of the present invention.These include
Compatible pharmaceutically active material, such as antipruritic, astringent, local anesthetic or antiphlogistic and other use
To improve material (such as coloring agent, preservative, antioxidant, opacifier, the thickening of formulation physical property
Agent and stabilizer etc.).It can in addition contain add some auxiliary reagents, such as lubricant, preservative, stabilizer,
Wetting agent, emulsifying agent, the salt for affecting osmotic pressure, buffer solution, colouring agent and aromatic substance etc..Certainly,
The addition of these auxiliary substances should not interfere with the activity and using effect of nano particle of the present invention.If any need
Will, preparation should carry out sterilization treatment after preparing.
The pharmaceutical composition of the present invention can serve as antistaphylohemolysin reagent.The staphylococcus includes but does not limit
In：MRSE (Staphylococcus epidermidis), staphylococcus aureus, more preferably methoxy west
Woods resistant Staphylococcus aureus.
The pharmaceutical composition of the present invention can especially pass through to reduce on mammal (such as the mankind) skin or skin
The staphylococcus quantity of survival is treating staphy lococcus infection in skin.The invention provides treatment staphylococcus,
For example infection of staphylococcus aureus and reduce survival aureus cell quantity method.These
Method includes the mammal that the nano particle of the pharmaceutical composition or the present invention of the present invention is applied to infection
Skin on.The quantity of the staphylococcus aureus of survival is reduced and can be proved by following feature：By golden yellow
The skin injury that color staphylococcus causes mitigates, the skin of the mammal by carrying staphylococcus aureus
Wipe staphylococcus aureus on piece to fall the reduction of quantity, or Staphylococcus aureus can be measured by any other
The appropriate method of bacterium number amount is verified.
Quantity except reducing active golden yellow color aureus cell, the pharmaceutical composition or nanometer of the present invention
Grain can also mitigate the symptom of infection of staphylococcus aureus.
The pharmaceutical composition of the present invention can make various formulations, including but not limited to：Suitable systemic applications
Formulation or the formulation of external application or local administration.
In a particular embodiment, the formulation is included but is not limited to：Tablet, solution, suspension,
Capsule, granule, pulvis, injection, patch, spray, ointment, ointment, cream, gel
Agent, creme, drops, spray, lotion.For example, the formulation includes the formulation of external application or local administration,
Including but not limited to：Patch, spray, ointment, ointment, cream, gel, creme, drops,
Spray, lotion.
The pharmaceutical composition of the present invention can also include other antibiotic, so as to be combined with other antibiotic, as long as this
A little other Conventional antibiotics are with nano particle of the present invention not to producing harmful effect each other.With alone Conventional antibiotic phase
Than it is beneficial that consumption, reduction toxic and side effect and the raising therapeutic effect of reduction Conventional antibiotic etc. are played in above-mentioned combination
Effect.Described other antibiotic can treat staphylococcus, preferred staphylococcus aureus (Staphylococcus
aureus)；More preferably methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus
Aureus the antibiotic for) infecting, including but not limited to：Vancomycin, cynnematin, Linezolid, impersonate drawing
Rather, Arbekacin, quinupristin, Dalfopristin, quinoline Knoop fourth, clindamycin, Daptomycin, Li Fu
It is flat, for drawing the medicines such as ten thousand stars, Tetracyclines such as tigecycline.It should also be understood by those skilled in the art that described
Antibiotic can also be the medicine for treating other microorganism infections such that it is able to treat different microorganism infections
Or the microorganism infection of complexity, for example, the antibiotic can be quinolones, beta-lactam, big ring
Lactone, aminoglycosides, amphenicols, nitro glyoxaline etc..
Additionally, those skilled in the art should know, others discussed above antibiotic can using with the present invention
Nano particle identical method of administration, give in same administration time；Can also be using the nanometer with the present invention
The different method of administration of grain, give in same administration time；Can also be using the nano particle identical with the present invention
Method of administration, give in different administration times.For example, above-mentioned other antibiotic can be with the nanometer of the present invention
Grain is discretely present in same pharmaceutical composition (for example, in kit), such that it is able to using identical or different
Method of administration, give in identical or different administration time.
In a preferred embodiment, pharmaceutical composition of the invention is aqueous pharmaceutical compositions, i.e. do not contained
The pharmaceutical composition of machine solvent.Those skilled in the art can teaching of the invention and actual demand decision medicine
The concentration of aliphatic acid in composition.
Advantages of the present invention：
1. the non-vesicle type nano particle of the present invention possesses significant antibacterial activity, and specificity for golden yellow
Color staphylococcus；
2. composition of the non-vesicle type nano particle of the present invention from natural origin, safe, has no toxic side effect；
3. the non-vesicle type nano particle of the present invention possesses excellent stability；
4. the non-vesicle type nano particle of the present invention avoids using organic solvents such as DMSO delivering medicine
Thing；
5. the preparation method of non-vesicle type nano particle of the invention is simple, has without using chloroform etc. is poisonous and hazardous
Machine solvent, so as to reduce production cost and environmentally friendly；
6. non-vesicle type nano particle diameter of the invention is little, is more easy to play bactericidal action into organization internal；
7. non-vesicle type nano particle polydispersity of the invention is little, and homogeneity is good, stable sterilization effect.
Unless stated otherwise, all technical terms and scientific terminology used in the present invention are all of the invention affiliated
What the those of ordinary skill of technical field was generally understood.Although the present invention can be used and be illustrated with this patent
Similar or equivalent method implement with material, but hereafter still elaborate suitable method and material.This
All publications, patent application, patent and other references being previously mentioned in patent are once reference i.e. full text
Include.Conflict as these publications, patent application, patent and other references are had with present patent application,
It is defined by this specification (including in being defined on).Additionally, material, method and example in present patent application are only
With explaining, it is not restricted.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for
The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (Cold Spring
Harbor Laboratory Press, 2001) described in condition, or according to the condition proposed by manufacturer.
Unless otherwise indicated, otherwise percentage and number are calculated by weight.
Embodiment
Material and method
Material：Egg yolk lecithin (egg PC), cholesterol, C6-NBD phytosphingosines (C6NBD) and 1,2-
Two myristoyls-sn- glyceryls -3- phosphoethanolamines-N- lissamine rhodamines B sulfonyls (DMPE-RhB) are purchased
From Avanti Polar Lipids Co., Ltds (Alabaster, AL)；Leukotrienes, trypticase soya broth
(TSB), phosphate buffer salt (PBS), trifluoroacetic acid (TFA), acetonitrile and sephadex G -75 are purchased from Sigma
Aldrich(St.Louis,MO)；Agar is purchased from BD (sparks, MD).
The culture of bacterium：MRSA252 bacterial strains (available from ATCC) are taken out from frost storage, in 37 DEG C of pancreas eggs
Overnight incubation on white enzyme soy agar plate.Following one independent bacterium colony is at tryptic soy broth (TSB)
Inoculation, and cultivate at 37 DEG C, rock 0.7 or so (logarithmic growth ranks are reached until the OD600 of culture medium
Section).Then bacterium is obtained after 3 minutes by 4000*g is centrifuged, using aseptic PBS twice afterwards.When
After removal PBS is centrifuged, the bacterium of acquisition is used for later in being suspended in the TSB of proper amount of fresh.
The preparation of nano particle of the present invention and sign：By 200mg surfactants (polysorbas20 or Tween 80)
With lipid (egg yolk lecithin:Cholesterol=9:1 weight ratio) mixture be suspended in the water of 4mL, wherein table
Face activating agent：The ratio of liposome is respectively 10:0、8:2、5:5、3:7、1:9 or 0:10.Suspension is stirred
Mix until formed homogeneous phase solution, be then heated to more than the fusing point of surfactant and lipid (20 DEG C, 30 DEG C,
40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C or 80 DEG C, the surfactant and lipid and its ratio depending on using).
During the leukotrienes (0.1%w/v to 4%w/v) of respective concentration is injected towards suspension and stir 30 minutes.Hereafter
Solution room temperature left overnight.The hydrodynamic size of nano particle of the present invention, uses Malvern Zetasizer
ZS instruments (Malvern instrument company of Britain, Britain) are measured.The average diameter of nano particle of the present invention is to pass through
Dynamic light scattering (DLS) is determined.All of property measurement is repeated 3 times at 25 DEG C.Comprising the linolenic present invention
Nano particle is herein also referred to as " TNAN-2 ".
The stability of nano particle of the present invention：The long-time stability of nano particle of the present invention (include 1%w/v's
Leukotrienes) by by nano-solution in 4 DEG C, the 25 DEG C or 37 DEG C storages time span of 3 months investigating.Every
Individual predetermined time point, measures the particle diameter of sample to judge nano particle of the present invention at different temperatures
Stability.
The antibacterial activity in vitro of nano particle of the present invention：In order to determine nano particle of the present invention to MRSA252
External MIC, by the MRSA252 (1*10 of fixed dosage6CFU, 200 μ L) add 2mL variable concentrations
In nano particle test tube solution of the present invention (0-0.6%w/v is in PBS), and rock at 37 DEG C.It is little through 5
When or the cultivation of 24 hours, measurement sample is in the trap (optical density, OD600 at 600nm) at 600nm.
All of detection all operation repetitives three times.
Measure of the measure of external MBC values similar to MIC.By the MRSA252 (1*10 of fixed dosage6
CFU, 200 μ L) add 2mL variable concentrations nanoparticles solution of the present invention in (0-0.6%w/v, PBS
In), and rock at 37 DEG C.After the cultivation of 24 hours, mixing suspension is diluted to 1 with aseptic PBS:10
To 1:105Times, take 5 μ L suspension and be placed in observation, the then cultivation overnight at 37 DEG C on TSB agar plates.It
Quantify the CFU of record MRSA252 afterwards.All of measurement operation repetitive three times.
The morphology of the staphylococcus aureus that nano particle of the present invention is processed：By SEM
(SEM) measure the form of the nanoparticle treated or untreated MRSA of the Jing present invention.MRSA252 is thin
Bacterium is cultivated 24 hours with nano particle of the present invention.Bacterium is collected and passes through FEI XL30 environment scan electronics and shows
Micro mirror is imaged.Processing procedure before bacteria samples imaging is as follows：Processed with nano particle of the present invention and untreated
Bacterium be centrifuged and remove supernatant liquid, fix two hours, after immobilization at room temperature with 2% glutaraldehyde,
Sample is centrifuged off glutaraldehyde, and is resuspended in the deionized water of 100 μ L again.Afterwards, 5 μ L's is thin
Bacterium suspension is dripped in polished silicon slice and dry overnight in Biohazard Safety Equipment.With scanning after bacteria samples chromium plating
Electronic Speculum is observed.
Antibacterial activity in vivo of the nano particle of the present invention to MRSA252：This is assessed in following two models
Antibacterial activity in vivo and therapeutic effect that bright nano particle is infected to MRSA252：Mouse skin wound infection mould
Type and mouse subcutaneous infection model.
In order to set up epidermal wound model, mouse (from Charles River laboratories) is carried out ketamine
With xylazine lumbar injection, after anesthesia, the black hair of mouse is shaved off, and skin is cleaned by ethanol pad.
Skin abrasion wound is manufactured in mouse back epidermis, by specified 1*1cm2Region 28G pins
Head is divided into the cross spider of 6*6.The mode of these cut manufactures guarantees that they only scratch cuticula and epidermis upper strata,
But do not scratch corium.After scratching 5 minutes, 50 μ L contain 1 × 107The MRSA252 bacterium PBS suspension of CFU
The region of interaction scuffing is seeded in by micropipette.After inoculation 30 minutes, nano particle of the present invention
PBS gels are used for damage field.Gel used is that polyethylene glycol is by appropriate by hydroxyethyl cellulose, glycerine
The hydrogel of radio modulation.These medicines were smeared once in continuous 5 days daily.Blank suspension is coagulated
Glue is also smeared as control experiment.At the 6th day, mouse was carried out euthanasia；Skin histology passes through 8mm
The skin punctures machine bacterial number removing and included counted.
In order to set up subcutaneous infection model, the black hair of mouse is shaved off, and skin is cleaned by ethanol pad.It
Afterwards, the 1 × 10 of 20 μ L6The MRSA252 bacterium PBS suspension of CFU is subcutaneously injected into shaving region, after
And the nano particle of the present invention of 200 μ L is injected in same area.Aseptic PBS tests quilt as blank
Injection.(at infection) physiology presentation and form are examined.After microbionation 3 days, the group of wound is recorded
Knit analysis result.
Toxicity in vivo is studied：The dermal toxicity of nano particle of the present invention is tested with the skin of back of ICR mouse.
Specifically, the back of mouse before the study 24 hours by shaving.Afterwards within 7 days daily with the present invention
Nano particle gel is smeared once in shaving region.It is used as control group with the mouse that PBS is smeared.In order to avoid
Gel drying, (mouse) skin are covered with by gauze.Behind 24 hours of last time topical administration, mouse quilt
Implement euthanasia, skin is crosscutting as histological examination by the cross-sectional slice of 8mm.The skin group of every mouse
10% buffered formalin process 18 hours is woven in, is then inserted in paraffin.These histotomies H＆E
Method is dyeed.The apoptosis of epithelial cell is analyzed assessment with TUNEL methods.Section is then by Hamamatsu
NanoZoomer2.0HT (digital slices scanner) is imaged.Picture NDP image softwares process.Per group of reality
There are 5 mouse (n=5) in testing.In order to assess toxicity, tissue sample scores according to Draize systems.Points-scoring system
It is as follows：0- does not have irritant proof；1- is minimum, is almost difficult to discover；In 2- epidermal areas clearly, it is seen that
Stimulate；Serious stimulation in 3- epidermal areas；Seriously stimulate in 4- epidermal areas, stimulate along with skin corium；5-
The serious stimulation of epidermal area and skin corium.In order to analyze macrophages infiltration, the skin histology of mouse passes through 8
Mm cross-sectional slices are collected.The skin biopsy of these frosts, uses FITC- anti-mouses to skin macrophage
F4/80 antibody is dyeed, and nucleus is dyeed using DAPI.After dyeing, skin samples lead at once
Cross the imaging of Nikon Delta Macroview fluorescence microscopes.
The preparation of 1 nano particle of the present invention of embodiment and sign
Nano particle of the invention is prepared as " material and method " part is described.
The hydrodynamic particle size of nano particle of the present invention is described by two parameters：Z- average grain diameters and many
Monodispersity index, two parameters are got by the cumulant analytical calculation of dynamic light scattering measurement.It is logical
Change linolenic acid concentration is crossed, a series of nano particle of the present invention is produced and detects to judge most suitable formulation.
As shown in figure 1, when linolenic acid concentration is 1%w/v, solution is presented to be clarified and transparent (Fig. 1 d), therein
The average grain diameter of nano particle of the present invention be 10nm, and average 0.2 polydispersity index.Therefore, exist
The nano particle selected in subsequent experimental includes 1%w/v leukotrienes, the wherein quality of lipid and surfactant
Than for 2:1.
Additionally, the present inventor is also have detected when contained linolenic concentration is between 0.2%w/v to 0.9%w/v
Surface potential of the nano particle of the present invention in water is in -3mV～-6mV.
The antibacterial activity in vitro of the nano particle of 2. present invention of embodiment
The nano particle of the present invention is by checking when bacterium and difference to the antibacterial activity in vitro of MRSA252
Antibacterial and bactericidal action that the nano particle of the present invention of concentration occurs when educating altogether is assessing.Under study for action,
The concentrations of nanoparticles (MIC) of the present invention of minimum bacteria growing inhibiting is measured first.Specifically, 1 ×
106The nano particle of the present invention of the MRSA252 and 0-0.6%w/v concentration of CFU is educated altogether.Be no less than
The bacterium of the nano particle culture of the present invention of 0.1%w/v concentration keeps obvious clarified solution, illustrates that this is dense
Degree significantly inhibits effect (Fig. 3 A) to bacterial growth.By contrast, when the concentration of nano particle of the present invention is low
When 0.1%w/v, inoculum just becomes muddy, shows substantial amounts of bacterial growth.In order to quantify point
The growth of analysis bacterium, behind 5 hours or 24 hours for cultivating, mixed liquor needs measured OD600 to judge bacterium
(1OD600 correspond to 10 to quantity8CFU/mL).It is as shown in Fig. 3 B and C, dense when nano particle of the present invention
When degree is higher than 0.1%w/v, the growth of bacterium is suppressed.In sum, 0.1%w/v is nanometer of the present invention
MIC of the particle for MRSA252.
MBC (MBC) of the nano particle of the present invention to MRSA252 is have detected also.Specifically,
It is determined that MBC values be defined as killing the lowest concentration of antimicrobial of 99.9% target bacteria MRSA252.One
In individual experiment in vitro, nano particle of the present invention and the MRSA252 (1*10 of different concentration6CFU 24) are educated altogether
Hour.Such cultivation causes nano particle of the present invention to contact with MRSA252 and carry out sterilization.After cultivation,
5 μ L are taken from inoculum to put on a lbmc agar plate, the cultivation overnight at 37 DEG C then is then right
The CFU of MRSA252 is counted.Fig. 4 A are that the nano particle of the present invention of variable concentrations and bacterium liquid are educated altogether
The representative photo that 5 μ L are cultivated in agar plate is sampled after 24 hours.It is obvious that the drug concentration for using is higher,
It is fewer that agar plate is left visible bacterium colony.A group bacteria culture media every afterwards takes 5 μ L, carries out 1:10 to 1:105
Dilution, is placed on TSB agar plates afterwards and observes and measure CFU.As shown in Figure 4 B, 0.2%w/v this
Bright nano particle kills 99.9% MRSA252.When linolenic concentration is raised to more than 0.2%w/v, carefully
Bacterium is all dead.Therefore, 0.2%w/v is MBC value of the nano particle of the present invention for MRSA252.
Result above shows MBC of the nano particle of the present invention to MRSA252：MIC ratios are about 2:
1, show that this nano particle is bactericide to the bacterium.Meanwhile, this nano particle caused thin in 24 hours
Bacterium number amount reduces by 3 Logarithmic degrees, shows bactericidal action of this nano particle to MRSA.This research can be by
The concentration and Drug exposure time for carrying out interior evaluating plays directive function.
In order to have more deep understanding to the mechanism that nano particle of the present invention kills MRSA252, the present inventor enters
One step checked morphological change of the MRSA252 bacteriums after nano particle before processing of the present invention.In process
Before, SEM imagings represent that MRSA252 cells have a botryoidalis of typical 0.6-1 μ m diameters, accumulation shape,
With complete cell membrane/membrane structure (Fig. 5 A).Through the present invention nano particle process 24 hours after, SEM into
Substantial variation as showing ne ar, including the destruction of significant eucaryotic cell structure, irregular aggregation shape
State, and damaged fuzzy cell edges (Fig. 5 B).This morphologic change means nano particle of the present invention to bacterium
The strong devastating effect of cell, so as to bacteria growing inhibiting.
The storage stability of 3. nano particle of the present invention of embodiment
The present inventor has investigated the long-time stability of nano particle of the present invention at different temperatures 3 months.Surely
Qualitatively change by detecting particle diameter to embody.As shown in Figure 2, store at 4 DEG C, 25 DEG C and 37 DEG C
The particle diameter of nano particle only rises to 13nm from 11nm, and change is negligible, and indicates the present invention
High stability of the nano particle under all conditions of storage.
Used as medicinal, nano particle of the present invention is very crucial a little will to be maintained in the case where long-time is preserved
Effect bactericidal action, i.e. after it will have stable structure, will also have stable activity.It is this in order to assess
Ability, the present inventor checked MIC the and MBC values of the nano particle of the present invention within the storage time of 3 months.
As shown in fig. 6, after storing at room temperature three months, nano particle of the present invention shows and fresh preparation
The approximate MIC and MBC values of sample.
The internal antibiotic effect of 4. nano particle of the present invention of embodiment
Antibacterial activity in vivo that nano particle of the present invention is infected to MRSA252 and therapeutic effect by using
The skin abrasion infection model of ICR mouse is further evaluated.(mouse) infection by MRSA252 cause by
It is coated with the gel and Blank gel of endocrine nano particle in continuous 5 days daily.Bacterial loads are in MRSA252
Metainfective 6th day determined.Skin histology homogeneity first in PBS, and with MRSA specificity fine jade
Cultivation overnight is carried out on fat plate (sweet dew alkoxide agar).Being specifically chosen mannite agar plate carries out MRSA cultures
It is because that the color of agar can be as the growth of MRSA be from pink colour yellowing.As shown in Figure 7 A, the present invention
Nanoparticle treated MRSA agar plates holding pink colour, but those (using Blank gel) control groups
Agar plate becomes yellow.This observation means that nano particle of the present invention has powerful at skin abrasion infection
Bactericidal activity.In addition to visual observations, the present inventor has also further carried out count number to the CFU values of sample
Change.As a result show, the bacterium amount retained on the mouse skin that Blank gel was processed is nano particle of the present invention
About 66 times for processing, the assumed value of wherein t distribution inspections are less than 0.01 (Fig. 7 B).
In order to the anti-MRSA for preferably assessing nano particle of the present invention it is active, also in ICR mouse subcutaneous infections
Model in further tested the nano particle.During this investigation it turned out, after infecting 24 hours, it is blank
The mouse of gel for treating shows purulence damage at infection.The gel for treating of nano particle of the present invention it is little
Mouse at the infection near show fash, but the order of severity light (Fig. 8).It is after 72 hours, blank solidifying
The pathology of the mouse of glue treatment becomes readily apparent from, it is meant that the diffusion of mouse In vivo infection.By contrast,
The mouse of the gel for treating of nano particle of the present invention shows unconspicuous pathology.This relatively shows the present invention
The excellent anti-MRSA curative effects of nano particle.
Toxicity of 5. nano particle of the present invention of embodiment to normal skin tissue
By in 5 days by outside the gel preparations of nano particle of the present invention on the epidermis of shaving mouse testing
The toxicity of nano particle of the present invention.As shown in figure 9, not finding that nano particle of the present invention causes any erythema
Or oedema stimulates.
In order to further assess excitant and Apoptosis, H＆E dyeing is carried out and end deoxynucleotide has turned
Move enzyme mark (TUNEL) experiment.As shown in Figure 10, the nanoparticle treated skin surface of the present invention keeps
Not not destroyed structure, above skin corium, have one layer of clearly healthy epidermal cell, this and it is undressed
Dermatological specimens are the same.H＆E dyeing is also demonstrated that, compared with undressed skin, the present invention receives
Rice grain process does not cause inflammation in any tissue.In addition, also carried out TUNEL analyses to assess
The quantity that serious DNA destructions and non-viable non-apoptotic cell are generated is produced in skin histology.In Fig. 10, nanometer of the present invention
Compared with untreated skin, the skin histology that particle disposal is crossed does not show that obvious apoptosis dyeing increases.
In order to further verify the security of nano particle of the present invention, especially it is whether to cause scytitis, I
Measure the macrophage level of Cutaneous permeation.In here research, the Jing present invention is nanoparticle treated
With the skin macrophage in the skin histology of not processed mistake, contaminated with FITC- anti-mouse f4/80 antibody
Color.As shown in figure 11, can observe in the lower position of the epidermal area above skin corium that skin macrophage is thin
Born of the same parents, the process of nano particle of the present invention do not change such Tissue distribution.Compared with undressed skin
Sample, the nanoparticle treated sample of those present invention do not show the obvious increasing for going out to permeate very much macrophage
Plus, show no obvious inflammation.
Antibacterial action of 6. leukotrienes of embodiment in water-based system
The present inventor further tests the nano particle of the present invention in water-based system and free leukotrienes pin
Antibacterial action to MRSA, as a result as shown in figure 14.
As a result find, in water-based system (PBS), can be notable comprising linolenic nano particle of the present invention
Killing MRSA, and free leukotrienes almost no bactericidal action in water-based system.
The preparation of of the present invention nano particle of the embodiment 7. comprising different aliphatic acid and Antimicrobial Screening
The present inventor is repeated above-described embodiment 1-2, so as to being prepared for comprising palmitic acid and arachidonic receiving
Rice grain.The present inventor has further screened these bactericidal actions of the non-vesicle type nano particle to MRSA,
As a result it is as shown in figure 15.
As a result find, comprising palmitic acid and arachidonic nano particle for MRSA is not significantly killed
Bacterium acts on.
The all documents referred in the present invention are all incorporated as reference in this application, just as each document
It is individually recited such as reference.In addition, it is to be understood that after the above-mentioned instruction content for having read the present invention,
Those skilled in the art can be made various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (14)
1. a kind of non-vesicle type nano particle, the particle is by aliphatic acid, surfactant and optional lipid
Constitute, wherein, the aliphatic acid is C16-C20, the aliphatic acid of preferred C17-C19.
2. non-vesicle type nano particle as claimed in claim 1, it is characterised in that the aliphatic acid is leukotrienes.
3. non-vesicle type nano particle as claimed in claim 1 or 2, it is characterised in that the non-vesicle type is received
The particle diameter of rice grain is 1-90nm；It is preferred that 2-80nm；More preferably 5-50nm；More preferably 5-20nm；Most preferably
5-15nm。
4. non-vesicle type nano particle as claimed in claim 1 or 2, it is characterised in that the nano particle
Polydispersity coefficient is<0.3；It is preferred that<0.2.
5. non-vesicle type nano particle as claimed in claim 1 or 2, it is characterised in that the non-vesicle type is received
The stability of rice grain is：
After preserving 2 months under room temperature, compared with the nano particle of fresh preparation, the nano particle is directed to MRSA
" minimal inhibitory concentration (MIC) " and " MBC (MBC) " value change be less than 20%；
Preferably smaller than 10%；Or,
Change less than 20% in the particle diameter of 4 DEG C, the 25 DEG C and 37 DEG C storages nano particle of the present invention of 3 months；It is excellent
The particle diameter of anthology invention nano particle changes less than 15%；The particle diameter of nano particle more preferably of the present invention changes little
In 10%.
6. non-vesicle type nano particle as claimed in claim 1 or 2, it is characterised in that the nano particle by
Prepared by following methods, methods described includes：
1). by surfactant and optional lipid suspension in water；
2). the suspension that 1) stirring obtains is until form homogeneous suspension liquid；
3). it is more than the fusing point of surfactant and optional lipid contained by the homogeneous suspension liquid to its that 2) heating obtains；
4). aliphatic acid is added in the hot suspension for 3) obtaining and stirred；
5). cooling, the suspension for 4) obtaining is stood, so as to obtain the non-vesicle type nano granule suspension of the present invention.
7. the preparation method of the non-vesicle type nano particle any one of claim 1-5, including following step
Suddenly：
1). by surfactant and optional lipid suspension in water；
2). the suspension that 1) stirring obtains is until form homogeneous suspension liquid；
3). it is more than the fusing point of surfactant and optional lipid contained by the homogeneous suspension liquid to its that 2) heating obtains；
4). aliphatic acid is added in the hot suspension for 3) obtaining and stirred；
5). cooling, the suspension for 4) obtaining is stood, so as to obtain the non-vesica any one of claim 1-5
Type nano particle.
8. a kind of pharmaceutical composition, its include non-vesicle type nano particle any one of claim 1-6 with
And optional pharmaceutically acceptable carrier.
9. pharmaceutical composition as claimed in claim 8, it is characterised in that described pharmaceutical composition can also include which
Its antibiotic.
10. pharmaceutical composition as claimed in claim 8 or 9, it is characterised in that described pharmaceutical composition is aqueous
Pharmaceutical composition.
The medicine any one of nano particle or claim 8-10 any one of 11. claims 1-6
Purposes of the compositions in antistaphylohemolysin (Staphylococcus) reagent is prepared.
12. purposes as claimed in claim 11, it is characterised in that the staphylococcus is staphylococcus aureus
(Staphylococcus aureus)；More preferably methicillin-resistant staphylococcus aureus (Methicillin-resistant
Staphylococcus aureus)。
A kind of 13. treatment methods, the treatment method are included the nanometer any one of claim 1-6
Grain gives object to treat microorganism infection.
A kind of 14. treatment methods, the treatment method are included the nanometer any one of claim 1-6
Grain gives object to treat microorganism infection with other Antibiotic combinations.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510594042.7A CN106539758A (en) | 2015-09-17 | 2015-09-17 | A kind of non-vesicle type nano particle of high stable and its application in treatment infection of staphylococcus aureus |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101385714A (en) * | 2008-10-24 | 2009-03-18 | 苏州纳康生物科技有限公司 | Preparation method of DHA lipid nano-particles |
| CN102048696A (en) * | 2011-01-13 | 2011-05-11 | 南昌大学 | Preparation method of lipid nanoparticles |
-
2015
- 2015-09-17 CN CN201510594042.7A patent/CN106539758A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101385714A (en) * | 2008-10-24 | 2009-03-18 | 苏州纳康生物科技有限公司 | Preparation method of DHA lipid nano-particles |
| CN102048696A (en) * | 2011-01-13 | 2011-05-11 | 南昌大学 | Preparation method of lipid nanoparticles |
Non-Patent Citations (1)
| Title |
|---|
| SOUICHI OHTA,ET AL: "Antibiotic Acitivity of Unsaturated Fatty Acids on Methicillin-resistant Staphylococcus aureus", 《BIOSCIENCE,BIOTECHNOLOGY,BIOCHEMISTRY》 * |
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Inventor after: Tan Bin Inventor after: Wang Gang Inventor after: Liu Yan Inventor before: Tan Bin |
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Application publication date: 20170329 |